The present invention relates to compounds, their salts, processes for making them and their use in investigating the processing of amyloid protein precursor and by extension Alzheimer""s Disease.
Alzheimer""s Disease (AD) is characterised by the abnormal deposition of amyloid in the brain in the form of extra-cellular plaques and intra-cellular neurofibrillary tangles. The rate of amyloid accumulation is a combination of the rates of formation, aggregation and egress from the brain. It is generally accepted that the main constituent of amyloid plaques is the 4 kD amyloid protein (xcex2A4, also referred to as Axcex2, xcex2-protein and xcex2AP) which is a proteolytic product of a precursor protein of much larger size. The ragged NH2- and COOH-termini of the native Axcex2 amyloid indicates that a complex mechanism of proteolysis is involved in its biogenesis.
The amyloid precursor protein (APP or Axcex2PP) has a receptor-like structure with a large ectodomain, a membrane spanning region and a short cytoplasmic tail. Different isoforms of APP result from the alternative splicing of three exons in a single gene and have 695, 751 and 770 amino acids respectively.
The Axcex2 domain encompasses parts of both extra-cellular and transmembrane domains of APP, thus its release implies the existence of two distinct proteolytic events to generate its NH2- and COOH-termini. At least two secretory mechanisms exist which release APP from the membrane and generate the soluble, COOH-truncated forms of APP (APPs). Proteases which release APP and its fragments from the membrane are termed xe2x80x9csecretasesxe2x80x9d. Most APPs is released by a putative xcex1-secretase which cleaves within the Axcex2 domain (between residues Lys16 and Leu17) to release xcex1-APP, and precludes the release of intact Axcex2. A minor portion of APPs is released by a xcex2-secretase, which cleaves near the NH2-terminus of Axcex2 and produces COOH-terminal fragments (CTFs) which contain the whole Axcex2 domain. Finding these fragments in the extracellular compartment suggests that another proteolytic activity (xcex3-secretase) exists under normal conditions which can generate the COOH-terminus of Axcex2.
It is believed that xcex3-secretase itself depends for its activity on the presence of presenilin-1. In a manner that is not fully understood presenilin-1 appears to undergo autocleavage.
The present compounds are useful for investigating amyloidosis in particular in aiding the isolation and characterization of proteases involved in processing APP, especially where those proteases are xcex3-secretase and/or presenilin-1.
Accordingly, the present invention provides a compound of formula I or a salt thereof: 
wherein:
R1 is benzoylphenyl or benzoylphenylC1-6alkyl wherein the benzoylphenyl moiety is optionally substituted by from one to nine bromine atoms and the alkyl moiety is optionally substituted by C1-6alkylsulfonylamino; or C1-6alkoxy;
R2 and R3 are independently chosen from C1-10alkyl, C1-10alkoxy, C2-10alkenyl, C2 ioalkenyloxy, C2-10alkynyl or C2-10alkynyloxy; phenyl; naphthyl; a five-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; a six-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms; and a group (CH2)pQ1 wherein Q1 is phenyl, naphthyl, a five-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S, and a six-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms; and wherein each of R2 and R3 is independently optionally substituted by one to three groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-3alkyl, C2-3 alkenyl and C2-3alkynyl,
(d) C1-3alkoxy,
(e) NR6R7 wherein R6 and R7 are independently chosen from hydrogen, C1-5alkyl and C1-5alkoxyC1-5alkyl,
(f) CO2R8 wherein R8 is hydrogen or C1-4alkyl,
(g) CONR6R7 or OCONR6R7 wherein R6 and R7 are independently as defined above,
(h) SO2NR6R7 wherein R6 and R7 are independently as defined above,
(i) CH2NR6R7 wherein R6 and R7 are independently as defined above,
(j) N(R8)COR8xe2x80x2 wherein R8 is independently as defined above and R8xe2x80x2 is independently as defined for R8,
(k) NR8SO2R8xe2x80x2 where R8 and R8xe2x80x2 are independently as defined above;
alternatively R3 may be hydrogen;
R4 and R5 are independently chosen from hydrogen, C1-6alkyl optionally substituted by halogen, hydroxy, thiol, amino, C1-4alkoxy, C1-4 alkylthio, carboxy, C1-4 alkoxycarbonyl and (CH2)qQ2 wherein Q2 is a five-membered unsaturated heterocycle containing 1, 2, 3 or 4 heteroatom optionally chosen from O, N, and S providing that not more than one heteroatom is O or S, a six-membered unsaturated heterocycle containing 1, 2 or 3 N atoms and phenyl and naphthyl, or a fused ring which is indolyl, each of the foregoing rings being optionally substituted with one to three groups independently chosen from hydroxy, C1-4alkyl, C1-4alkoxy, thiol, C1-4alkylthio, halogen, amino, carboxy, amido, CO2H and xe2x80x94NHC(NH2)2 and wherein each of the foregoing rings is optionally fused to a benzene ring;
alternatively R5 may be benzoylbenzyl which is optionally substituted by from one to nine bromine atoms;
B is Cxe2x95x90O or CHOH in the R configuration;
L is a bond or [(CH2)mNHCO]n in which one of the methylene groups may be replaced by a disulphide group;
Z is (CH2)kamino, benzoxy or biotin, or when L is a bond then Z is hydrogen or biotin providing that when Z is hydrogen then either R1 is not C1-6alkoxy or R5 is benzoylbenzyl;
k is an integer of from one to ten;
each m is independently an integer of from one to ten;
n is an integer of from one to ten;
p is zero, one, two or three; and
q is zero, one, two or three;
with the proviso that no carbon atom is substituted by more than one hydroxy group.
In an embodiment the compounds of the present invention are of formula Ixe2x80x2: 
where R1, R2, R3, R4, R5, L and Z are as defined above.
In one embodiment the compounds of the present invention are of formula Ixe2x80x3: 
where R1, R2, R3, R4, R5, L and Z are as defined above.
In another embodiment there are provided compounds of formula Ixe2x80x2xe2x80x3: 
where R1, R2, R3, R4, R5, L and Z are as defined above.
The following preferred definitions of substituents apply to each of the formulae: I, Ixe2x80x2, Ixe2x80x3 and Ixe2x80x2xe2x80x3 which refer to those substituents.
Preferably R1 is tertiarybutoxy or benzoylphenyl or benzoylphenylC1-2alkyl wherein the benzoylphenyl moiety is optionally substituted by from one to six, preferably by from one to four, bromine atoms, and the C1-2alkyl moiety is optionally substituted by methylsulfonylamino.
Particular values of R1 are tertiarybutoxy, benzoylphenyl, 2,3,5,6-tetrabromobenzoylphenyl, benzoylphenylethyl, (1-methylsulfonylamino)benzoylphenylethyl, benzoylphenylmethyl and 4-bromobenzoylphenyl.
R2 and R3 may be independently chosen from phenyl; naphthyl; a five-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; a six-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms; and a group (CH2)pQ1 wherein Q1 is phenyl; naphthyl; a five-membered heteroaromatic ring containing 1, 2, 3 or 4 heteroatoms independently chosen from O, N and S, at most one of the heteroatoms being O or S; and a six-membered heteroaromatic ring containing 1, 2 or 3 nitrogen atoms; and wherein each of R2 and R3 is independently optionally substituted by one to three groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-3alkyl, C2-3alkenyl and C2-3alkynyl,
(d) C1-3alkoxy,
(e) NR6R7 wherein R6 and R7 are independently as defined above,
(f) CO2R8 wherein R8 is independently as defined above,
(g) CONR6R7 wherein R6 and R7 are independently as defined above,
(h) SO2NR6R7 wherein R6 and R7 are independently as defined above,
(i) CH2NR6R7 wherein R6 and R7 are independently as defined above,
(j) N(R8)COR8xe2x80x2 wherein R8 and R8xe2x80x2 are independently as defined above,
(k) NR8SO2R8xe2x80x2 where R8 and R8xe2x80x2 are independently as defined above;
More preferably R2 and R3 are (CH2)pQ1.
Preferably Q1 is phenyl optionally substituted by one or two groups independently chosen from:
(a) halogen, cyano and nitro,
(b) hydroxy,
(c) C1-4alkyl, C2-4alkenyl and C2-4alkynyl,
(d) C1-4alkoxy and
(e) amino.
In one embodiment R2 and R3 are both benzyl.
More preferably Q1 is phenyl.
R2 and R3 are especially benzyl.
Preferably R4 and R5 are independently chosen from optionally substituted C1-6alkyl and (CH2)qQ2. More preferably R4 and R5 are independently chosen from C1-6alkyl and (CH2)qQ2.
Preferably Q2 is optionally substituted phenyl. More preferably Q2 is phenyl.
In particular R4 and R5 are independently chosen from methyl, benzyl, phenyl, 2-methylpropyl, 1-hydroxyethyl and isobutyl. R4 is particularly isobutyl.
R5 is optionally benzoylbenzyl optionally substituted by from one to six bromine atoms, particularly by from one to four bromine atoms. R5 may be unsubstituted benzoylbenzyl or benzyl.
L is preferably [(CH2)mNHCO]n. Particular values of L-Z are biotincarbonylaminonpentyl, amino, biotincarbonylaminonpentylcarbonylaminonpentyl, botincarbonylaminonpentylcarbonylaminonpentylcarbonylaminonpentyl, biotincarbonylaminonpentylcarbonylaminonpentylcarbonylaminonpentylearbon ylaminonpentyl, blotincarbonylaminoethyldlsulphidethylcarbonylaminonpentylcarbonylaminonpentylcarbonylaminonpentylcarbonylaminonpentyl, benzoxycarbonylaminonpentylcarbonylaminonpentyl, benzoxycarbonylaminonpentylcarbonylaminonpentylcarbonylaminonpentylcarb onylaminonpentyl and aminonbutylcarbonylaminonpentylcarbonylaminonpentylcarbonylaminonpentyl.
Z is preferably biotin.
k is preferably an integer from three to five, such as four.
m is preferably an integer of from two to seven, more preferably from three to six, especially five.
n is preferably an integer of from one to seven, particularly from one to five.
p is preferably one.
q is preferably zero or one.
Thus a subclass of compounds is provided wherein:
R1 is tertiarybutoxy, benzoylphenyl or benzoylphenylC1-2alkyl wherein the benzoylphenyl moiety is optionally substituted by from one to four bromine atoms and the C1-2alkyl moiety is optionally substituted by methylsulfonylamino;
R2 and R3 are benzyl;
R4 is isobutyl;
R5 is benzyl or benzoylbenzyl;
B is CHOH in the R configuration;
L is a bond or [(CH2)mNHCO]n in which one of the methylene groups may be replaced by a disulphide group;
Z is (CH2)kamino, benzoxy or biotin, or when L is a bond then Z is hydrogen providing either R1 is not tertiary butoxy or R5 is not benzyl;
k is four;
m is five; and
n is an integer of from one to five.
For the avoidance of doubt each time the moieties R6, R7, R8 and R8xe2x80x2 occur they are chosen independently.
Also for the avoidance of doubt, radiolabelled compounds are encompassed within the above formulae. Thus, for example, hydrogen atoms may be replaced by tritium atoms.
For the avoidance of doubt xe2x80x9cbiotinxe2x80x9d in the above definitions means the residue left when CO2H is removed from commercially available biotin.
As used herein, the expression xe2x80x9cC1-10alkylxe2x80x9d includes methyl and ethyl groups, and straight-chained and branched propyl, butyl, pentyl and hexyl groups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl and t-butyl. Derived expressions such as xe2x80x9cC1-6alkylxe2x80x9d, xe2x80x9cC1-4alkylxe2x80x9d, xe2x80x9cC2-10alkenylxe2x80x9d, xe2x80x9cC2-4alkenylxe2x80x9d, xe2x80x9cC2-10alkynyl and xe2x80x9cC2-4alkynylxe2x80x9d are to be construed in an analogous manner.
The expression xe2x80x9cC3-7cycloalkylxe2x80x9d as used herein includes cyclic propyl, butyl, pentyl, hexyl and heptyl groups such as cyclopropyl and cyclohexyl.
The term xe2x80x9cheterocyclicxe2x80x9d includes rings which are saturated, partially saturated or unsaturated. Unsaturated heterocyclic rings are also known as beteroaromatic rings.
Suitable 5- and 6-membered heteroaromatic rings include pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, oxadiazolyl and thiadiazolyl groups. A suitable 5-membered heteroaromatic ring containing four nitrogen atoms is tetrazolyl. Suitable 6-membered heteroaromatic rings containing three nitrogen atoms include 1,2,4-triazine and 1,3,5-triazine. Suitable saturated heterocyclic rings include piperazine, morpholine, piperidine, tetrahydrofuran and tetrahydrothiophene. Tetrahydrofuran is preferred.
The term xe2x80x9chalogenxe2x80x9d as used herein includes fluorine, chlorine, bromine and iodine, of which fluorine and chlorine are preferred.
As used herein the term xe2x80x9cC1-6alkoxyxe2x80x9d includes methoxy and ethoxy groups, and straight-chained, branched and cyclic propoxy and butoxy groups, including cyclopropylmethoxy.
Specific Examples according to the present invention include:
{4R-[1S-(2-(4-benzoyl-phenyl)-1-{5-[5-(2-oxo-(3aR,6aS)hexahydro-thieno[3,4-d]imidazol-6S-yl)-pentanoylamino]-pentylcarbamoyl}-ethylcarbamoyl)-3-(1S)-methyl-butylcarbamoyl]-1S-benzyl-2R-hydroxy-5-phenyl-pentyl}-carbamic acid tert-butyl ester;
4-benzoyl-N-{4R-[1S-(2-(4-benzoyl-phenyl)-1-{5-[5-(2-oxo-(3aR,6aS)hexahydro-thieno[3,4-d]imidazol-6S-yl)-pentanoylamino]-pentylcarbamoyl}-ethylcarbamoyl)-3(1S)-methyl-butylcarbamoyl]-1S-benzyl-2R-hydroxy-5-phenyl-pentyl}-benzamide;
(4R-{1S-[2-(4-benzoyl-phenyl)-1-carbamoyl-ethylcarbamoyl]-3(1S)-methyl-butylcarbamoyl}-1S-benzyl-2R-hydroxy-5-phenyl-pentyl)-carbamic acid tert-butyl ester;
4-benzoyl-N-{1S-benzyl-4R-[1-(1-carbamoyl-2S-phenyl-ethylcarbamoyl)-3(1S)-methyl-butylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl}-benzamide;
4-benzoyl-N-{1S-benzyl-4R-[-(1-carbamoyl-2S-phenyl-ethylcarbamoyl)-3(1S)-methyl-butylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl}-2,3,5,6-tetrabromo-benzamide;
5S-[3-(4-benzoyl-phenyl)-propionylamino]-2R-benzyl-4R-hydroxy-6-phenyl-hexanoic acid [1-(1-carbamoyl-2S-phenyl-ethylcarbamoyl)-3(1S)-methyl-butyl]-amide;
5S-[3-(4-benzoyl-phenyl)-2S-methanesulfonylamino-propionylamino]-2R-benzyl-4R-hydroxy-6-phenyl-hexanoic acid [1-(1-carbamoyl-2S-phenyl-ethylcarbamoyl)-3(1S)-methyl-butyl]-amide;
N-{1S-benzyl-4R-[1-(1-carbamoyl-2S-phenyl-ethylcarbamoyl)-3(1S)-methyl-butylcarbamoyl]-2R-hydroxy-5-phenyl-pentyl}-4-(4-bromo-benzoyl)-benzamide;
4-benzoyl-N-{1S-benzyl-2R-hydroxy-4R-[3-methyl-1S-(1-{5-[5-(2-oxo-(3aR,6aS)hexahydro-thieno[3,4-d]imidazol-6S-yl)-pentanoylamino]-pentylcarbamoyl}-2S-phenyl-ethylcarbamoyl)-butylcarbamoyl]-5-phenyl-pentyl}-benzamide;
{1S-benzyl-2R-hydroxy-4R-[3(1S)-methyl-1-(1-{5-[5-(2-oxo-(3aR,6aS)hexahydro-thieno[3,4-d]imidazol-6S-yl)-pentanoylamino]-pentylcarbamoyl}-2S-phenyl-ethylcarbamoyl)-butylcarbamoyl]-5-phenyl-pentyl}-carbamic acid tert-butyl ester;
(1S-benzyl-2R-hydroxy-4R-{3(1S)-methyl-1-[1-(5-{6-[5-(2-oxo(3aR,6aS)-hexahydro-thieno[3,4-d]imidazol-6S-yl)-pentanoylamino]-hexanoylamino}-pentylcarbamoyl)-2S-phenyl-ethylcarbamoyl]-butylcarbamoyl}-5-phenyl-pentyl)-carbamic acid tert-bytyl ester;
[1S-benzyl-2R-hydroxy-4R-(3(1S)-methyl-1-{1-[5-(6-{6-[5-(2-oxo(3aR,6aS)-hexahydro-thieno[3,4-d]imidazol-6S-yl)-pentanoylamino]-hexanoylamino}-hexanoylamino)-pentylcarbamoyl]-2S-phenyl-ethylcarbamoyl}-butylcarbamoyl)-5-phenyl-pentyl]-carbamic acid tert-butyl ester;
[1S-benzyl-2R-hydroxy-4R-(3(1S)-methyl-1-{1-[5-(6-(6-{6-[5-(2-oxo(3aR,6aS)-hexahydro-thieno[3,4-d]imidazol-6S-yl)-pentanoylamino]-hexanoylamino}-hexanoylamino)-hexanoylamino)-pentylcarbamoyl]-2S-phenyl-ethylcarbamoyl}-butylcarbamoyl)-5-phenyl-pentyl]-carbamic acid tert-butyl ester;
[1S-benzyl-4R-(1-{1-[5-(6-benzyloxycarbonylamino-hexanoylamino)-pentylcarbamoyl]-2S-phenyl-ethylcarbamoyl}-3(1S)-methyl-butylcarbamoyl)-2R-hydroxy-5 -phenyl-pentyl]-carbamic acid tert-butyl ester;
(1S-benzyl-4R-{1-[1-(5-{6-[6-(6-benzyloxycarbonylamino-hexanoylamino)-hexanoylamino]-hexanoylamino}-pentylcarbamoyl)-2S-phenyl-ethylcarbamoyl]-3(1S)-methyl-butylcarbamoyl}-2R-hydroxy-5-phenyl-pentyl)-carbamicacid tert-butyl ester;
(4R-{1S-[1-(5-{6-[6-(6-amino-hexanoylamino)-hexanoylamino]-hexanoylamino}-pentylcarbamoyl)-2-phenyl-ethylcarbamoyl]-3(1S)-methyl-butylcarbamoyl}-1S-benzyl-2R-hydroxy-5-phenyl-pentyl)-carbamic acid tert-butyl ester and
(1S,2R,4R,7S,10S)-[52-(hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl)-2-hydroxy-7-(2-methylpropyl)-5,8,11,19,26,33,40,48-octaoxo-1,4,10-tris(phenylmethyl)-43,44-dithia-6,9,12,18,25,32,39,47-octaazadopentacont-1-yl]carbamic acid 1,1-dimethylester
The compounds of the present invention have an activity as inhibitors of xcex3 secretase. In a preferred embodiment the compounds of the invention inhibit proteolysis of PS-1. The compounds can photoaffinity label the PS-1 beterodimer.
The present invention also provides a compound of the invention or a salt thereof for use in a method of investigating APP processing in amyloidosis.
There is also provided a process for producing a compound of formula I or a pharmaceutically acceptable salt thereof which comprises reacting a compound of formula II with a compound of formula III: 
wherein R1, R2, R3, R4 and R5 are as defined above, X is OH, C1-6alkoxy or NHLZ where L and Z are as defined above and P is hydrogen or a protecting group such as a trialkylsilane group, for example t-butyl dimethylsilyl, followed, if necessary, by deprotection of the resulting compound and, if necessary by converting X into a group NHLZ. The reaction is generally carried out in the presence of coupling agents such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole, generally both at 1.2 equivalents, in a solvent such as DMF, generally at about room temperature for six to twelve hours. Any necessary deprotection is achieved by conventional means. Conversion of X into a group NH2 can be achieved by, for example, reacting with N-methylmorpholine and isobutylchloroformate, both generally at 1 equivalent, followed by the addition of ammonia gas.
The compound of formula II is produced by reacting a compound of formula IV: 
wherein R1, R2 and R3 are as defined above in a solvent such as dioxane, with a base such as lithium hydroxide in a polar solvent such as water generally at room temperature for above five hours. If desired the resulting compound of formula II in which P is hydrogen is protected by conventional means.
The compound of formula IV is produced by reacting a compound of formula V with a compound of formula VI or a compound of formula VII: 
wherein R1, R2 and X are as defined above, R3xe2x80x3 is the acyl derivative of a group R3 as defined above and R3xe2x80x3 is a group R3 bound to an oxo group at the portion of R3 which connects to the compound of formula V. The reaction is generally carried out in the presence of a base such as lithium dilsopropylamide in a solvent such as THF generally cooled to xe2x88x9278xc2x0 C. for about thirty minutes. The reaction mixture is subsequently dehydrated without purification and then hydrogenated with, for example, hydrogen over 5% Pd/C catalyst at about 50 psi for about 2 h.
The compound of formula V in which R1 is tertiary butyl and R2 is benzyl can be prepared as described by J. Litera et al., Collect. Czech. Chem. Commun. 1998, 63, 231ff. Compounds of formula V in which R1 is other than tertiary butyl and R2 is other than benzyl can be made by analogous methods.
Compounds of formula III in which X is C1-6alkoxy can be made by reacting a compound of formula XIII with a compound of formula VIII: 
wherein R4 and R5 are as defined above and Pxe2x80x2 is a protecting group such as BOC, generally under the same conditions as for the reaction between compounds of formulae II and III. The compound of formula XIII is generally pre-reacted with HCl in C1-6alcohol to produce the C1-6alkyl ester hydrochloride salt. Compounds of formula III in which X is NH2 can be made using the reaction at the end of the description for making compounds of formula I.
Compounds of formula III in which X is Lxe2x80x94Z where L is not a bond and Z is as defined above can be made by reacting a compound of formula III in which X is OH and the free amine group is optionally protected, with a compound of formula IX:
H2NLZxe2x80x83xe2x80x83(IX)
in which L is not a bond and Z is as defined above, generally under the same conditions as for the reaction between compounds of formulae II and III.
Compounds of formula IX can be made by reacting a compound of formula X with a compound of formula XI:
H2NLxe2x80x2Wxe2x80x83xe2x80x83(X)
SuOCOLxe2x80x3Zxe2x80x83xe2x80x83(XI)
where Lxe2x80x2 is [(CH2)mxe2x80x2NHCO]nxe2x80x2, Lxe2x80x3 is [(CH2)mxe2x80x3NHCO]n-nxe2x80x2 in which one of the methylene groups may be replaced by a disulphide group, mxe2x80x2 and nxe2x80x2 are as defined above, each mxe2x80x3 is an integer from 1 to 10, nxe2x80x2 is an integer from 1 to 9, W is C1-6alkoxy, Z is as defined above and SuO is succinimidoxy optionally substituted at the 3-position with a group NaSO3, generally with 1 equivalent of each and in the presence of a strong base such as triethylamine. The product obtained is deprotected, for example with TFA.
It will be understood that any compound of formula I initially obtained from the above process may, where appropriate, subsequently be elaborated into a further compound of formula I by techniques known from the art. For example, the nature of R1 can be altered by reacting a compound of formula I in which the hydroxy group is protected, for example by a trialkylsilane group, and from which the RIO group has been removed, for example with TFA, with an appropriate acid or acid chloride generally under the same conditions as the reaction between compounds of formulae II and III.
The nature of R1 in compounds of formula IV can be altered by analogous methods.
As a further example the L in Lxe2x80x94Z of a compound of formula I can be lengthened where Z is benzoxy by hydrogenating, for example with 10% Pd/C under H2 at pressure and in ethanol, and coupling the resulting amine with a compound of formula XII or XIV:
HOCOLxe2x80x2xe2x80x3Zxe2x80x83xe2x80x83(XII)
SuOCOLxe2x80x2xe2x80x3Zxe2x80x83xe2x80x83(XIV)
where Lxe2x80x2xe2x80x3 is [(CH2)mxe2x80x2xe2x80x3NHCO]nxe2x80x2xe2x80x3 where mxe2x80x2xe2x80x3 and nxe2x80x2xe2x80x3 are as defined above for m and n but are no greater than nine and one of the methylene groups maybe replaced by a disulphide group and SuO and Z are as defined above, generally under the same conditions used for reacting the compounds of formulae II and III.
L in compounds of formula III can be lengthened by analogous methods.
Compounds of formulae VI, VII, VIII, X, XI, XII, XIII and XIV are commercially available or known in the prior art or can be made from commercially available or known compounds by standard methods.
It will also be appreciated that where more than one isomer can be obtained from a reaction then the resulting mixture of isomers can be separated by conventional means.
Where the above-described process for the preparation of the compounds according to the invention gives rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques such as preparative chromatography. The novel compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution.
During any of the above synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protective Groups in Organic Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
A typical assay which can be used to determine the level of activity of compounds of the present invention is as follows:
(1) Mouse neuroblastoma neuro 2a cells expressing human app695 are cultured at 50-70% confluency in the presence of sterile 10 mM sodium butyrate.
(2) Cells are placed in 96-well plates at 30,000/well/100 xcexcL in minimal essential medium (MEM) (phenol red-free)+10% foetal bovine serum (FBS), 50 mM HEPES buffer (pH7.3), 1% glutamine, 0.2mg/mL G418 antibiotic, 10 mM sodium butyrate.
(3) Make dilutions of the compound plate. Dilute stock solution to 5.5% DMSO/110 xcexcM compound. Mix compounds vigorously and store at 4xc2x0 C. until use.
(4) Add 10 xcexcL compound/well. Mix plate briefly, and leave for 18 h in 37xc2x0 C. incubator.
(5) Remove 90 xcexcL of culture supernatant and dilute 1:1 with ice-cold 25 mM HEPES (pH.3), 0.1% BSA, 1.0 mM EDTA (+broad spectrum protease inhibitor cocktail; pre-aliquotted into a 96-well plate). Mix and keep on ice or freeze at xe2x88x9280xc2x0 C.
(6) Add back 100 xcexcL of warm MEM+10% FBS, 50 mM HEPES (pH7.3), 1% glutamine, 0.2 mg/mL G418, 10 mM sodium butyrate to each well, and return plate to 37xc2x0 C. incubator.
(7) Prepare reagents necessary to determine amyloid peptide levels, for example by ELISA assay
(8) To determine if compounds are cytotoxic cell viability following compound administration is assessed by the use of redox dye reduction. A typical example is a combination of redox dye MTS (Promega) and the electron coupling reagent PES. This mixture is made up according to the manufacturer""s instructions and left at room temperature.
(9) Quantitate amyloid beta 40 and 42 peptides using an appropriate volume of diluted culture medium by standard ELISA techniques.
(10) Add 15 xcexcL/well MTS/PES solution to the cells; mix and leave at 37xc2x0 C.
(11) Read plate when the absorbance values are approximately 1.0 (mix briefly before reading to disperse the reduced formazan product).
The Examples of the present invention all had an ED50 of less than 500 nM, preferably less than 200 nM and most preferably less than 100 nM in the above assay.
The following examples illustrate the present invention.
The following General Procedures were used throughout the Examples.
A solution of an amine component, an acid component, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (1.2 equivalents) and 1-hydroxybenzotriazole (1.2 equivalents) in DMF was stirred at room temperature overnight. If the amine component was present as a salt, one equivalent of triethylamine was added. The reaction mixture was diluted with ethyl acetate and washed with an aqueous solution of citric acid (10%) (twice), aqueous NaHCO3 solution (twice) and brine. The organic phase was dried (MgSO4), filtered and evaporated in vacuo. The crude product was either used without further purification, or purified by trituration, crystallization or flash column chromatography.
A solution of an ester in dioxane was treated with aqueous lithium hydroxide (3 equivalents, 1.0 M in H2O) and stirred at room temperature. The reaction mixture was acidified with aqueous HCl and ethyl acetate. The organic layer was washed with brine, dried (MgSO4), filtered and evaporated in vacuo. The crude product was either used directly without further purification, or purified by trituration or flash column chromatography.
A BOC-protected amine was dissolved in trifluoroacetic acid and stirred under nitrogen at room temperature. The reaction mixture was evaporated in vacuo and azeotroped with toluene (twice). The crude product was either used directly or purified by flash column chromatography.
A silyl ether was dissolved in an excess of a solution of TBAF in THF (1.0 M) and stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate and aqueous citric acid. The resulting precipitate was collected by filtration and washed with water and ether (several times). The crude product was either used directly without further purification, or purified by column chromatography. In cases where a precipitate was not formed, the reaction mixture was diluted with warm ethyl acetate and washed with dilute citric acid and brine. The organic phase was dried (MgSO4), filtered and evaporated in vacuo. Purification by flash column chromatography gave the deprotected alcohol.
An amine and an N-hydroxysuccinimide ester (1 equivalent) were dissolved in DMF and treated with triethylamine (1 equivalent) and stirred at room temperature for 1 h. The reaction mixture was diluted with ethyl acetate and washed with an aqueous solution of citric acid (10%) (twice), aqueous NaHCO3 solution (twice) and brine. The organic phase was dried (MgSO4), filtered and evaporated in vacuo. The crude product was either used without further purification, or purified by trituration or flash column chromatography.